The present invention relates to an image data producing method and apparatus and, particularly, to such an image data producing method and apparatus which can produce low-noise image data rapidly with a simple operation by irradiating an image carrier including independently formed and two-dimensionally distributed specimen spots, at least some of which contain a fluorescent substance, with a stimulating ray to excite the fluorescent substance and photoelectrically detecting fluorescence emission released from the fluorescent substance.
An autoradiographic system using as a detecting material for detecting radiation a stimulable phosphor which can absorb, store and record the energy of radiation when it is irradiated with radiation and which, when it is then stimulated by an electromagnetic wave having a specified wavelength, can release stimulated emission whose light amount corresponds to the amount of radiation with which it was irradiated is known, which comprises the steps of introducing a radioactive labeling substance into an organism, using the organism or a part of the tissue of the organism as a specimen, superposing the specimen and a stimulable phosphor sheet formed with a stimulable phosphor layer for a certain period of time, storing and recording radiation energy in a stimulable phosphor contained in the stimulable phosphor layer, scanning the stimulable phosphor layer with an electromagnetic wave to excite the stimulable phosphor, photoelectrically detecting the stimulated emission released from the stimulable phosphor to produce digital image signals, effecting image processing on the obtained digital image signals, and reproducing an image on displaying means such as a CRT or the like or a photographic film (see, for example, Japanese Patent Publication No. 1-60784, Japanese Patent Publication No. 1-60782, Japanese Patent Publication No. 4-3952 and the like).
Unlike the system using a photographic film, according to the autoradiographic system using the stimulable phosphor as a detecting material, development, which is chemical processing, becomes unnecessary. Further, it is possible reproduce a desired image by effecting image processing on the obtained image data and effect quantitative analysis using a computer. Use of a stimulable phosphor in these processes is therefore advantageous.
On the other hand, a fluorescence detecting system using a fluorescent substance as a labeling substance instead of a radioactive labeling substance in the autoradiographic system is known. According to this system, it is possible to study a genetic sequence, to study the expression level of a gene, and to effect separation or identification of protein or estimation of the molecular weight or properties of protein or the like. For example, this system can perform a process including the steps of distributing a plurality of DNA fragments on a gel support by means of electrophoresis after a fluorescent dye was added to a solution containing a plurality of DNA fragments to be distributed, or distributing a plurality of DNA fragments on a gel support containing a fluorescent dye, or dipping a gel support on which a plurality of DNA fragments have been distributed by means of electrophoresis in a solution containing a fluorescent dye, thereby labeling the electrophoresed DNA fragments, exciting the fluorescent dye by a stimulating ray to cause it to release fluorescent light, detecting the released fluorescent light to produce an image and detecting the distribution of the DNA fragments on the gel support. This system can also perform a process including the steps of distributing a plurality of DNA fragments on a gel support by means of electrophoresis, denaturing the DNA fragments, transferring at least a part of the denatured DNA fragments onto a transfer support such as a nitrocellulose support by the Southern-blotting method, hybridizing a probe prepared by labeling target DNA and DNA or RNA complementary thereto with the denatured DNA fragments, thereby selectively labeling only the DNA fragments complementary to the probe DNA or probe RNA, exciting the fluorescent dye by a stimulating ray to cause it to release fluorescent light, detecting the released fluorescent light to produce an image and detecting the distribution of the target DNA on the transfer support. This system can further perform a process including the steps of preparing a DNA probe complementary to DNA containing a target gene labeled by a labeling substance, hybridizing it with DNA on a transfer support, combining an enzyme with the complementary DNA labeled by a labeling substance, causing the enzyme to contact a fluorescent substance, transforming the fluorescent substance to a fluorescent substance having fluorescent light releasing property, exciting the thus produced fluorescent substance by a stimulating ray to release fluorescent light, detecting the fluorescent light to produce an image and detecting the distribution of the target DNA on the transfer support. This fluorescence detecting system is advantageous in that a genetic sequence or the like can be easily detected without using a radioactive substance.
Furthermore, an analysis plate, called a micro-titer plate, that is provided with numerous wells in which a specimen solution can be held is known to be used with one fluorescence detecting system. According to this system, biochemical analysis is effected by accommodating a specimen solution labeled with a fluorescent substance in wells, irradiating the specimen solution with a stimulating ray to excite the fluorescent substance and detecting fluorescence emission released from the fluorescent substance.
Further, a micro-array detecting system has been recently developed, which comprises the steps of using a spotting device to drop at different positions on the surface of a carrier such as a slide glass plate, a membrane filter or the like specific binding substances, which can specifically bind with a substance derived from a living organism such as a hormone, tumor marker, enzyme, antibody, antigen, abzyme, other protein, a nuclear acid, cDNA, DNA, RNA or the like and whose sequence, base length, composition and the like are known, thereby forming a number of independent spots, specifically binding the specific binding substances using a hybridization method or the like with a substance derived from a living organism such as a hormone, tumor marker, enzyme, antibody, antigen, abzyme, other protein, a nuclear acid, cDNA, DNA or mRNA, which is gathered from a living organism by extraction, isolation or the like or is further subjected to chemical processing, chemical modification or the like and which is labeled with a labeling substance such as a fluorescent substance, dye or the like, thereby forming a micro-array, irradiating the micro-array with a stimulating ray, photoelectrically detecting light such as fluorescence emitted from a labeling substance such as a fluorescent substance, dye or the like, and analyzing the substance derived from a living organism. This micro-array image detecting system is advantageous in that a substance derived from a living organism can be analyzed in a short time period by forming a number of spots of specific binding substances at different positions of the surface of a carrier such as a slide glass plate at high density and hybridizing them with a substance derived from a living organism and labeled with a labeling substance.
In the fluorescent detecting system and the micro-array detecting system, data for biochemical analysis such as image data are produced by irradiating a labeling substance with a stimulating ray to excite it and photoelectrically detecting fluorescence emission released from the labeling substance by a light detector. Therefore, since noise is generated to lower the accuracy of analysis if the stimulating ray enters the light detector, a stimulating ray cut filter is provided for blocking the stimulating ray and preventing it from entering the light detector.
However, even when a stimulating ray cut filter is provided, it is difficult to completely block the stimulating ray. Therefore, it has been proposed to lower noise caused by detecting the stimulating ray by stopping the irradiation with the stimulating ray after irradiating a labeling substance with the stimulating ray to excite the labeling substance and detecting residual fluorescence emission released from the labeling substance even after the completion of the irradiation with the stimulating ray.
In the case of using this method, residual fluorescence emission is detected by irradiating a fluorescent substance contained in a specimen solution held in a number of wells of a micro-titer plate, biochemical analysis data are conventionally produced by sequentially irradiating the wells with a stimulating ray to excite the fluorescent substance contained in the specimen solution in the wells and residual fluorescence emission released from the fluorescent substance after stopping the irradiation with the stimulating ray is photoelectrically detected using a light detector such as a photomultiplier.
However, since the micro-titer plate is formed with numerous wells, e.g., 96 wells, and the amount of residual fluorescence emission detected by one-time irradiation with the stimulating ray is small, it is necessary to repeat an operation comprising the steps of irradiation with the stimulating ray, stopping the irradiation with the stimulating ray and detecting residual fluorescence emission. Therefore, it inevitably takes a long time to irradiate the fluorescent substance contained in the specimen solution in all wells, photoelectrically detect residual fluorescence emission substance using a light detector such as a photomultiplier and produce biochemical analysis data. As a result, the progress of reaction of the specimen solution in the wells becomes different between the individual wells.
In order to solve this problem, biochemical analysis data are conventionally produced by stimulating the fluorescent substance contained in a specimen solution in a well and photoelectrically detecting residual fluorescence emission from the substance using a light detector such as a photomultiplier, each time the specimen solution is poured into a well.
However, it is not only troublesome to produce biochemical analysis data in this manner but also time-consuming and uneconomical.
In the micro-array detecting system, the state of spots obtained by spotting specific binding substances onto the surface of a substrate such as a slide glass plate or a membrane filter and hybridizing a substance derived from a living organism and labeled with a labeling substance with them may change with the elapse of time and, therefore, there may arise similar problems to those in the micro-titer plate.
It is therefore an object of the present invention to provide an image data producing method and apparatus which can produce low-noise image data rapidly with a simple operation by irradiating an image carrier including independently formed and two-dimensionally distributed specimen spots, at least some of which contain a fluorescent substance, with a stimulating ray to excite the fluorescent substance and photoelectrically detecting fluorescence emission released from the fluorescent substance.
The above and other objects of the present invention can be accomplished by an image data producing method comprising the steps of irradiating an image carrier including independently formed and two-dimensionally distributed specimen spots, at least some of which contain a fluorescent substance, with a stimulating ray to excite the fluorescent substance contained in the specimens, stopping the irradiation of the image carrier with the stimulating ray, and photoelectrically detecting residual fluorescence emission released from the fluorescent substance contained in the specimens after stopping the irradiation with the stimulating ray using a two-dimensional area sensor.
According to the present invention, since image data are produced by irradiating an image carrier with a stimulating ray to excite the fluorescent substance contained in the specimens, stopping the irradiation of the image carrier with the stimulating ray, and photoelectrically detecting residual fluorescence emission released from the fluorescent substance contained in the specimens after stopping the irradiation with the stimulating ray using a two-dimensional area sensor, the image carrier is not irradiated with the stimulating ray while the two-dimensional area sensor detects the residual fluorescence emission and, therefore, it is possible to markedly lower noise in image data caused by detecting the stimulating ray by the two-dimensional area sensor.
In a preferred aspect of the present invention, the steps of irradiating the image carrier with the stimulating ray to excite the fluorescent substance contained in the specimens, stopping the irradiation of the image carrier with the stimulating ray, and photoelectrically detecting residual fluorescence emission released from the fluorescent substance contained in the specimens after stopping the irradiation with the stimulating ray using the two-dimensional area sensor are repeated two or more times.
According to this preferred aspect of the present invention, since the steps of irradiating the image carrier with the stimulating ray to excite the fluorescent substance contained in the specimens, stopping the irradiation of the image carrier with the stimulating ray, and photoelectrically detecting residual fluorescence emission released from the fluorescent substance contained in the specimens after stopping the irradiation with the stimulating ray using the two-dimensional area sensor are repeated two or more times, a sufficient amount of the residual fluorescence emission can be detected by the two-dimensional area sensor and, therefore, image data capable of generating a desired image can be produced.
In a further preferred aspect of the present invention, the steps of irradiating the image carrier with the stimulating ray to excite the fluorescent substance contained in the specimens, stopping the irradiation of the image carrier with the stimulating ray, and photoelectrically detecting residual fluorescence emission released from the fluorescent substance contained in the specimens after stopping the irradiation with the stimulating ray using the two-dimensional area sensor are performed by synchronizing on and off operations of at least one stimulating ray source for emitting a stimulating ray and opening and closing operations of a shutter of the two-dimensional area sensor.
According to this preferred aspect of the present invention, since the steps of irradiating the image carrier with the stimulating ray to excite the fluorescent substance contained in the specimens, stopping the irradiation of the image carrier with the stimulating ray, and photoelectrically detecting residual fluorescence emission released from the fluorescent substance contained in the specimens after stopping the irradiation with the stimulating ray using the two-dimensional area sensor are performed by synchronizing on and off operations of at least one stimulating ray source for emitting a stimulating ray and opening and closing operations of a shutter of the two-dimensional area sensor, residual fluorescence emission can be reliably detected by the two-dimensional area sensor only when the image carrier is not irradiated with the stimulating ray and, therefore, it is possible to markedly lower noise in image data caused by detecting the stimulating ray by the two-dimensional area sensor.
In another preferred aspect of the present invention, the steps of irradiating the image carrier with the stimulating ray to excite the fluorescent substance contained in the specimens, stopping the irradiation of the image carrier with the stimulating ray, and photoelectrically detecting residual fluorescence emission released from the fluorescent substance contained in the specimens after stopping the irradiation with the stimulating ray using the two-dimensional area sensor are performed by synchronizing on-off operations of at least one stimulating ray source for emitting a stimulating ray and opening and closing operations of a shutter of the two-dimensional area sensor using a chopper.
According to this preferred aspect of the present invention, since the steps of irradiating the image carrier with the stimulating ray to excite the fluorescent substance contained in the specimens, stopping the irradiation of the image carrier with the stimulating ray, and photoelectrically detecting residual fluorescence emission released from the fluorescent substance contained in the specimens after stopping the irradiation with the stimulating ray using the two-dimensional area sensor are performed by synchronizing on-off operations of at least one stimulating ray source for emitting a stimulating ray and opening and closing operations of a shutter of the two-dimensional area sensor using a chopper, residual fluorescence emission can be reliably detected by the two-dimensional area sensor only when the image carrier is not irradiated with the stimulating ray and, therefore, it is possible to markedly lower noise in image data caused by detecting the stimulating ray by the two-dimensional area sensor.
In a further preferred aspect of the present invention, image data are produced by detecting residual fluorescence emission by the two-dimensional area sensor via a filter for cutting at least light having a wavelength of the stimulating ray.
According to this preferred aspect of the present invention, since residual fluorescence emission is detected by the two-dimensional area sensor via the filter for cutting at least light having a wavelength of the stimulating ray, even if the stimulating ray is present for some reason in an apparatus when residual fluorescence emission is detected by the two-dimensional area sensor, it is possible to reliably prevent the stimulating ray from being detected by the two-dimensional area sensor and, therefore, it is possible to markedly lower noise in image data caused by detecting the stimulating ray by the two-dimensional area sensor.
In a further preferred aspect of the present invention, image data are produced by detecting residual fluorescence emission by the two-dimensional area sensor via a Fresnel lens.
According to this preferred aspect of the present invention, since residual fluorescence emission is detected by the two-dimensional area sensor via the Fresnel lens, in the case where an image of fluorescent dye labeling a specimen solution contained in a number of wells of the microtiter plate is read out, image data can be produced without parallax by detecting fluorescence emission released from the micro-titer plate by the two-dimensional area sensor.
In a further preferred aspect of the present invention, image data are produced by using a CCD camera as the two-dimensional area sensor.
In a further preferred aspect of the present invention, image data are produced by using a cooled CCD camera as the two-dimensional area sensor.
According to this preferred aspect of the present invention, since a cooled CCD camera is used as the two-dimensional area sensor, it is possible to expose the cooled CCD camera for a sufficient long time period and, therefore, even if the residual fluorescence emission is very weak, image data can be produced in a desired manner.
In a further preferred aspect of the present invention, a micro-titer plate formed with numerous wells holding specimens labeled with a fluorescent dye is used as the image carrier.
According to this preferred aspect of the present invention, since image data of the fluorescent dye labeling specimens held in all wells formed in the micro-titer plate can be produced by the two-dimensional area sensor at the same time, it is possible for all wells formed in the micro-titer plate to hold specimens labeled with the fluorescent dye at one time and, therefore, it is possible to produce image data with markedly low noise rapidly with a simple operation.
The above other objects of the present invention can be accomplished by an image data producing apparatus comprising at least one stimulating ray source for emitting a stimulating ray, a stage on which an image carrier including independently formed and two-dimensionally distributed specimen spots, at least some of which contain a fluorescent substance, is to be placed, a two-dimensional area sensor, and a control means for irradiating the image carrier placed on the stage with a stimulating ray emitted from the at least one stimulating ray source, thereby exciting a fluorescent substance contained in the specimens, stopping the irradiation with the stimulating ray and causing the two-dimensional area sensor to photoelectrically detect residual fluorescence emission released from the fluorescent substance contained in the specimens.
According to the present invention, since the image data producing apparatus includes the control means for irradiating the image carrier placed on the stage with a stimulating ray emitted from the at least one stimulating ray source, thereby exciting a fluorescent substance contained in the specimens, stopping the irradiation with the stimulating ray and causing the two-dimensional area sensor to photoelectrically detect residual fluorescence emission released from the fluorescent substance contained in the specimens, the image carrier is not irradiated with the stimulating ray while the two-dimensional area sensor detects residual fluorescence emission and, therefore, it is possible to markedly lower noise in image data caused by detecting the stimulating ray by the two-dimensional area sensor.
In a preferred aspect of the present invention, the control means is constituted so as to repeat the steps of irradiating the image carrier placed on the stage with a stimulating ray emitted from the at least one stimulating ray source, thereby exciting a fluorescent substance contained in the specimens, stopping the irradiation with the stimulating ray and causing the two-dimensional area sensor to photoelectrically detect residual fluorescence emission released from the fluorescent substance contained in the specimens two or more times.
According to this preferred aspect of the present invention, since the steps of irradiating the image carrier placed on the stage with a stimulating ray emitted from the at least one stimulating ray source, thereby exciting a fluorescent substance contained in the specimens, stopping the irradiation with the stimulating ray and causing the two-dimensional area sensor to photoelectrically detect residual fluorescence emission released from the fluorescent substance contained in the specimens are repeated two or more times, a sufficient amount of the residual fluorescence emission can be detected by the two-dimensional area sensor and, therefore, image data capable of generating a desired image can be produced.
In a further preferred aspect of the present invention, the control means is constituted so as to synchronize on and off operations of at least one stimulating ray source and opening and closing operations of a shutter of the two-dimensional area sensor in such a manner that the shutter of the two-dimensional area sensor is closed when the at least one stimulating ray source is held on and it is opened only when the at least one stimulating ray source is held off, thereby irradiating the image carrier placed on the stage with a stimulating ray emitted from the at least one stimulating ray source to excite a fluorescent substance contained in the specimens, stopping the irradiation with the stimulating ray and causing the two-dimensional area sensor to photoelectrically detect residual fluorescence emission released from the fluorescent substance contained in the specimens.
According to this preferred aspect of the present invention, since the control means is constituted so as to synchronize on and off operations of at least one stimulating ray source and opening and closing operations of a shutter of the two-dimensional area sensor in such a manner that the shutter of the two-dimensional area sensor is closed when the at least one stimulating ray source is held on and is opened only when the at least one stimulating ray source is held off, thereby irradiating the image carrier placed on the stage with a stimulating ray emitted from the at least one stimulating ray source to excite a fluorescent substance contained in the specimens, stopping the irradiation with the stimulating ray and causing the two-dimensional area sensor to photoelectrically detect residual fluorescence emission released from the fluorescent substance contained in the specimens, residual fluorescence emission can be reliably detected by the two-dimensional area sensor only when the image carrier is not irradiated with the stimulating ray and, therefore, it is possible to markedly lower noise in image data caused by detecting the stimulating ray by the two-dimensional area sensor.
In another preferred aspect of the present invention, the image data producing apparatus further comprises a disk-like rotatable chopper having at least two openings and the control means is constituted so as to control the rotation of the chopper in such a manner that a light detecting surface of the two-dimensional area sensor is covered by the chopper when one of the openings of the chopper is located in an optical path of the stimulating ray emitted from the at least one stimulating ray source and one of the openings of the chopper is located in front of the light detecting surface of the two-dimensional area sensor only when the chopper is located in the optical path of the stimulating ray emitted from the at least one stimulating ray source to cut the stimulating ray.
According to this preferred aspect of the present invention, since the image data producing apparatus further comprises a disk-like rotatable chopper having at least two openings and the control means is constituted so as to control the rotation of the chopper in such a manner that a light detecting surface of the two-dimensional area sensor is covered by the chopper when one of the openings of the chopper is located in an optical path of the stimulating ray emitted from the at least one stimulating ray source and one of the openings of the chopper is located in front of the light detecting surface of the two-dimensional area sensor only when the chopper is located in the optical path of the stimulating ray emitted from the at least one stimulating ray source to cut the stimulating ray, residual fluorescence emission can be reliably detected by the two-dimensional area sensor only when the image carrier is not irradiated with the stimulating ray and, therefore, it is possible to markedly lower noise in image data caused by detecting the stimulating ray by the two-dimensional area sensor.
In a further preferred aspect of the present invention, the image data producing apparatus further comprises a light guide for leading a stimulating ray emitted from the at least one stimulating ray source and the control means is constituted so as to control the rotation of the chopper in such a manner that a light detecting surface of the two-dimensional area sensor is covered by the chopper when one of the openings of the chopper is located in front of a stimulating ray emitting end portion of the light guide and one of the openings of the chopper is located in front of the light detecting surface of the two-dimensional area sensor only when the chopper is located in front of the stimulating ray emitting end portion of the light guide to cut the stimulating ray.
According to this preferred aspect of the present invention, since the image data producing apparatus further comprises a light guide for leading a stimulating ray emitted from the at least one stimulating ray source, it is possible to efficiently irradiate the image carrier with a stimulating ray emitted from the at least one stimulating ray source.
In a further preferred aspect of the present invention, the light guide is constituted by an optical fiber bundle.
In a further preferred aspect of the present invention, the chopper is formed with four openings.
According to this preferred aspect of the present invention, even when the decay time of residual fluorescence emission released from a fluorescent substance is short, the residual fluorescence emission can be reliably detected by the two-dimensional area sensor without rotating the chopper at an excessively high speed only when the image carrier is not irradiated with the stimulating ray and, therefore, it is possible to markedly lower noise in image data caused by detecting the stimulating ray by the two-dimensional area sensor.
In a further preferred aspect of the present invention, the image data producing apparatus further comprises a filter for cutting at least light having a wavelength of the stimulating ray.
According to this preferred aspect of the present invention, since the image data producing apparatus further comprises a filter for cutting at least light having a wavelength of the stimulating ray, even if the stimulating ray is present for some reason in an apparatus when residual fluorescence emission is detected by the two-dimensional area sensor, it is possible to reliably prevent the stimulating ray from being detected by the two-dimensional area sensor and, therefore, it is possible to markedly lower noise in image data caused by detecting the stimulating ray by the two-dimensional area sensor.
In a further preferred aspect of the present invention, the image data producing apparatus further comprises a Fresnel lens between the stage and the two-dimensional area sensor.
According to this preferred aspect of the present invention, since the image data producing apparatus further comprises a Fresnel lens between the stage and the two-dimensional area sensor, in the case where an image of fluorescent dye labeling a specimen solution contained in a number of wells of the micro-titer plate is read out, image data can be produced without parallax by detecting fluorescence emission released from the micro-titer plate by the two-dimensional area sensor.
In a further preferred aspect of the present invention, the two-dimensional area sensor is constituted as a CCD camera.
In a further preferred aspect of the present invention, the two-dimensional area sensor is constituted as a cooled CCD camera.
According to this preferred aspect of the present invention, since the two-dimensional area sensor is constituted as a cooled CCD camera, it is possible to expose the cooled CCD camera for a sufficient long time period and, therefore, even if the residual fluorescence emission is very weak, image data can be produced in a desired manner.
In a further preferred aspect of the present invention, the image carrier is constituted by a micro-titer plate formed with numerous wells holding specimens labeled with a fluorescent dye.
According to this preferred aspect of the present invention, since image data of the fluorescent dye labeling specimens held in all wells formed in the micro-titer plate can be produced by the two-dimensional area sensor at the same time, it is possible for all wells formed in the micro-titer plate to hold specimens labeled with the fluorescent dye at one time and, therefore, it is possible to produce image data with markedly low noise rapidly with a simple operation.
The above and other objects and features of the present invention will become apparent from the following description made with reference to the accompanying drawings.